The fate of B cells is highly dependent on signals from the B cell antigen receptor (BCR) for development, tolerance, and activation decisions. The overall goal of this project is to understand the role of cholesterol- rich subdomains of the plasma membrane called lipid rafts in BCR signal transduction. Recent work in B cells has established that lipid rafts play a role in BCR signaling. Lipid rafts exist primarily as small, highly dynamic structures that coalesce into larger and more stable lipid rafts upon cell stimulation. BCR stimulation leads to its movement from the non-lipid raft plasma membrane into lipid rafts and induces coalescence of lipid rafts, eventually leading to a large lipid raft patch or cap on the cell surface. In Specific Aim 1, the mechanism of BCR-induced lipid raft coalescence will be studied. BCR stimulation causes ezrin to become dephosphorylated and to release its connection between some lipid raft proteins and the actin cytoskeleton. We shall test if this release promotes lipid raft coalescence and BCR signaling. We hypothesize that the later stages of lipid raft coalescence require active processes involving myosins and will attempt to identify the myosin isoform responsible. BCR stimulation also leads to outgrowth of long filopdia- like projections ("cytonemes") and in Specific Aim 2, we shall determine the mechanism of outgrowth of these projections. We shall explore the role of the B144 protein, the Rac1 and Cdc42 GTPases, and myosin 10 and its close relatives. In Specific Aim 3, we shall examine the connection of lipid rafts to the ability of the BCR to activate the key transcription factor, NF-kB. Carmal, a key scaffold molecule for this signaling pathway, is localized to lipid rafts and it recruits other components of this pathway to lipid rafts following BCR stimulation. The importance of Carmal localization to lipid rafts and of lipid raft coalescence for activation of NF-kB will be determined. Finally, in Specific Aim 4, we shall examine lipid raft coalescence and NF-kB pathway activation in immature B cells and B cell lines' of immature phenotype, which appear to differ from mature B cells in these processes. . Lay Language Statement: The ability of the immune system to respond to infectious agents depends on the function of the antigen receptors of lymphocytes. This function appears to involve changes in a subdomain of the plasma membrane called lipid rafts and the mechanisms of these changes will be studied.